The epidermal growth factor receptor (EGFR) signaling pathway is a central regulator of cell growth and proliferation and modulates critical cell cycle regulatory molecules. This pathway has emerged as a promising target for cancer therapy. EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib (Tarceva) and gefitinib (Iressa), are approved for cancer treatment but have induced a clinical response in only a subgroup of patients. Therefore, EGFR-TKI's molecular mechanism of action needs to be better understood. My long-term goal is to elucidate the molecular mechanism of action of EGFR-TKI so that novel therapeutic approaches or diagnostic tools that are clinically relevant can be developed. I have recently shown that in vitro erlotinib sensitivity is partially dependent on the activity of cyclin-dependent kinase 2 (Cdk2), which is the most downstream kinase of the EGFR pathway that regulates the transition from the G1 phase to the S phase. The objective of this application is to determine which downstream molecules of the EGFR and non-EGFR signaling pathway predict the response to EGFR-TKIs. The central hypothesis of this proposal is that the effect of Cdk2 activity on EGFR-TKI-mediated cytotoxicity is regulated by downstream molecules of the cell signaling pathway, specifically ERK, p27, and PEA15. This hypothesis is based on the following observations. First, erlotinib inhibits the tyrosine kinase of EGFR in both erlotinib-sensitive and erlotinib-resistant breast cancer cells; however, erlotinib inhibits Cdk2 activity only in sensitive cells. These findings indicate that there is an abnormality in the EGFR signaling pathway in erlotinib-resistant cells. Second, phosphorylated ERK is downregulated and p27 is upregulated in erlotinib-sensitive cells, and the phosphorylation status of p27 affects its nuclear-cytoplasmic localization and expression level. Third, PEA15 sequesters ERK into the cytoplasm from the nucleus and reduces cell proliferation. I have designed three independent but interrelated specific aims to provide a comprehensive assessment of the downstream EGFR and non-EGFR signaling pathway in breast cancer cells treated with EGFR-TKI. Specific Aim 1. Establish how erlotinib regulates p27 to suppress Cdk2 activity in vitro. Specific Aim 2. Establish how PEA15 modulates erlotinib sensitivity in vitro and in vivo. Specific Aim 3. Establish in vivo biomarkers that predict erlotinib sensitivity. Relevance: This study will provide significant insight into the role of downstream molecules affected by EGFR-TKI. Our findings will improve the outcome of cancer patients by increasing EGFR-TKI efficacy and facilitating the selection of patients who may benefit from EGFR-TKI therapy.